In this study, we present evidence that a glycine transporter, GLYT1, is expressed in neurons and that it is associated with glutamatergic synapses. Despite the presence of GLYT1 mRNA in both glial cells and in glutamatergic neurons, previous studies have mainly localized GLYT1 immunoreactivity to glial cells in the caudal regions of the nervous system. However, using novel sequence specific antibodies, we have identified GLYT1 not only in glia, but also in neurons. The immunostaining of neuronal elements could best be appreciated in forebrain areas such as the neocortex or the hippocampus, and it was found in fibers, terminal boutons and in some dendrites. Double labeling confocal microscopy with the glutamatergic marker vGLUT1 revealed an enrichment of GLYT1 in a subpopulation of glutamatergic terminals. Moreover, through electron microscopy, we observed an enrichment of GLYT1 in both the presynaptic and the postsynaptic aspects of putative glutamatergic terminals that established asymmetric synapses. In addition, we demonstrated that GLYT1 was physically associated with the NMDA receptor in a biochemical assay. In conclusion, the close spatial association of GLYT1 and glutamatergic synapses strongly supports a role for this protein in neurotransmission mediated by NMDA receptors in the forebrain, and perhaps in other regions of the CNS.